We will identify, characterize, and study replication origins in the vicinity of X-linked PGK (PGK- 1), which, by translocation mapping, is known to be located near the X chromosome inactivation center. One major aim of these experiments is to test the hypothesis that replication timing, that is, late replication, is an important component mechanism of cell memory and X chromosome inactivation. This hypothesis is attractive because some studies have indicated that late replication is the only feature of X chromosome inactivation common from egg laying mammals to man, and, moreover, it is theoretically possible for the late replication of a chromosomal domain to be a somatically heritable feature. Yeast artificial chromosomes containing all or part of the PGK- 1 gene will be mapped and ordered. Then either a new ligation-mediated PCR (LMPCR) approach or published methods will be used to identifying the direction of fork movement. Switches in the direction of fork movement will allow identification of both replication origins and replication termini. Once replication origins and termini in the region near PGK-1 have been identified, they will be characterized in depth to determine differences between these elements on the active, early replicating X chromosome and the inactive, late replicating X chromosome. Included in these in depth studies will be protein footprint, and chromatin structure analysis by LMPCR.